HIV encodes a number of accessory genes not commonly found in other retroviruses. The purpose of this project is to investigate the biological and biochemical functions of the HIV accessory proteins Vif and Vpu and to understand their precise role in virus replication. One of our goals is to characterize cellular factors involved in Vif or Vpu function. From our studies on Vpu we expect to gain insights into general principles of protein degradation from the ER and into mechanisms involving late stages of virus production, in particular the involvement of ion channels in the secretory pathway. As a result of our experiments on Vif, we not only expect to gain insights into the function of this viral factor but we expect to learn more about the role of the cytoskeleton in virus replication. Finally, we hope our research will provide a basis for the assessment of viral proteins as potential antiviral targets.The vpu gene is unique to HIV-1 and encodes a small integral membrane protein. Vpu regulates virus release from the cell surface and degradation of CD4 in the endoplasmic reticulum. These two biological activities of Vpu are based on two independent and distinct molecular mechanisms that can be attributed to separable structural domains of Vpu. Vpu-regulated virus release is sensitive to changes in the transmembrane (TM) domain of Vpu and is correlated with an ion channel activity of Vpu. CD4 degradation, on the other hand, involves a direct interaction of the Vpu and CD4 cytoplasmic domains. We demonstrated in the past that Vpu-mediated CD4 degradation involves the ubiquitin-dependent proteasome pathway and requires an interaction with a novel cellular protein, betaTrCP. BetaTrCP was found to engage in ternary complexes with Vpu and CD4 and represents a direct link to Skp1p, a known component of E3 ubiquitin ligase complexes. Unlike normal cellular substrates of TrCP, such as the NF-kappaB inhibitor IkappaB, Vpu is not targeted for degradation by proteasomes. In fact, we found that Vpu has a dominant negative effect on cellular TrCP function by acting as a competitive inhibitor of TrCP. Expression of Vpu in HIV-infected T cells or in inducible HeLa cells inhibited TNF-alpha- induced degradation of IkappaB-alpha. Vpu did not inhibit TNF-mediated activation of the IkappaB kinase but instead interfered with the subsequent TrCP-dependent degradation of phosphorylated IkappaB-alpha. In virus-producing cells, NF-kappaB activity was significantly increased in the absence of Vpu. This was, at least in part, due to the activity of the accessory protein Vpr. Consistent with its effect on IkappaB-alpha, Vpu substantially repressed the HIV-induced activation of NF-kappaB. Direct analysis of Vpr-mediated activation of NF-kappaB activity in the presence or absence of Vpu in vitro revealed a pronounced inhibition by Vpu. These results suggest a novel pathway by which Vpu regulates the activity of Vpr and demonstrate for the first time that the mechanism by which Vpr activates NF-kappaB involves the function of TrCP. Since NF-kappaB is a key regulator of cytokine expression, it is likely to play a role in the perturbation of cytokine production observed in HIV-infected individuals.Vif is a 23-kDa basic protein, which has an important function in regulating infectivity of progeny virions. The biochemical mechanism of Vif function is obscure. We analyzed the role of Vif by studying its subcellular distribution by cell fractionation as well as confocal microscopy. We found that a substantial portion of intracellular Vif protein is associated with the cytoskeleton, specifically intermediate filaments. The association of Vif with intermediate filaments is specific and can result in the reorganization of the cytoskeletal network. In addition, we found that Vif is incorporated into virus particles where it associates with the core of virions. Mutational analysis of Vif suggests a role for a N-terminal domain in Vif for the interaction with vimentin. Preliminary evidence also points to an interaction of Vif with viral or cellular RNA, presumably involving a central domain in Vif. Deletion of this central domain in Vif severely affects its subcellular distribution and inhibits virion incorporation of Vif.